Human tissue-type plasminogen activator is a thrombolytic agent that activates plasminogen to plasmin at the site of a fibrin clot (1,2,3). Plasmin is subsequently responsible for the proteolytic degradation of fibrin; thus, human tissue-type plasminogen activator mediates the dissolution of blood clots and is consequently useful in the treatment of various thrombotic disorders.
When human tissue-type plasminogen activator is used in thrombolytic therapy, monitoring a number of plasma components can be useful to evaluate the modifications in the coagulation and fibrinolytic systems. These components included fibrinogen, fibrin(ogen) degradation products (FDPs), plasminogen, alpha-2-antiplasmin and immunoreactive human tissue-type plasminogen activator. In order to measure circulating amounts of these components, as well as human tissue-type plasminogen activator, as indicia important to determine an effective therapeutic dose of human tissue-type plasminogen activator, blood samples are drawn prior to and during human tissue-type plasminogen activator administration. In such blood samples, human tissue-type plasminogen activator may continue to activate plasminogen to plasmin in vitro, which in turn can lead to in vitro consumption of fibrinogen, plasminogen, alpha-2-antiplasmin, and the production of FDPs resulting from the proteolysis of fibrinogen by plasmin.
Free human tissue-type plasminogen activator forms complexes with protease inhibitors such as alpha-2-antiplasmin, alpha-1-antitrypsin, alpha-2-macroglobulin, Cl-esterase inhibitor and human tissue-type plasminogen activator fast acting inhibitor (4-7). These complexes might alter the immunoreactivity of human tissue-type plasminogen activator in various degrees, which could cause inaccurate measurement of its concentration in plasma.
To accurately determine fibrinolytic changes in vivo, subsequent proteolytic events by plasmin must be minimized in vitro. It was perceived that the most direct method for minimizing or eliminating these proteolytic events (artifacts) would be to selectively neutralize the proteolytic activity of human tissue-type plasminogen activator. This could be accomplished by the rapid introduction of an irreversible protease inhibitor. Several such inhibitors include various peptide chloromethyl ketones (8,9).
Therefore, it is an object of the present invention to identify, prepare and use during plasma collection a rapidly acting, soluble inhibitor of human tissue-type plasminogen activator that does not interfere in an assay monitoring the total fibrinolytic system.